CN102286098A - Production method of natural hirudin - Google Patents
Production method of natural hirudin Download PDFInfo
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- CN102286098A CN102286098A CN2011102374956A CN201110237495A CN102286098A CN 102286098 A CN102286098 A CN 102286098A CN 2011102374956 A CN2011102374956 A CN 2011102374956A CN 201110237495 A CN201110237495 A CN 201110237495A CN 102286098 A CN102286098 A CN 102286098A
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Abstract
The invention discloses a production method of natural hirudin. The production method is characterized by comprising the steps of extracting, acid depositing, heating, dehydrating and drying natural hirudin liquid, wherein the content of active ingredients of the obtained natural hirudin powder can be detected through Markwardt direct thrombin titration so that the quality is stable and the curative effect of the product is ensured. The method has the characteristics of simple process, high extraction ratio, low production cost, high product purity, long storage period and the like and can be used for providing safe and high-quality raw materials for foods, health-care foods, medicines or cosmetics.
Description
Technical field
The present invention relates to a kind of production method of extracting the effective active composition from animal body, particularly is the method for raw material production natural hirudin with the hirudinaria manillensis.
Background technology
The low molecular polypeptide that natural hirudin is made up of 65～66 amino acid, its molecular weight only is 7000 dalton, and very easily water-soluble and physiological saline is highly stable at normal temperatures, simple temperature raises (100 ℃ of water-baths), or pH change (1～13) does not all influence its activity.Natural hirudin is the strongest natural antithrombin material of being found in the world up to now, and thrombotic diseases such as human cardiovascular and cerebrovascular diseases especially cerebral thrombosis are had special efficacy, its prevention effect be any material can not compare.Correlative study simultaneously shows that also natural hirudin is a kind of direct thrombin inhibitors, and its anticoagulation is very single-minded, and the anti-freezing effect is stronger than heparin, and natural hirudin does not rely on the participation of AT III and HC when the anticoagulant enzymic activity.Natural hirudin is compared with other anti-freezing medicines such as heparin, its anti-freezing effect is more reliable, this be because: 1. natural hirudin does not combine with plasma proteins, the kind of plasma proteins, concentration do not influence its activity, the anti-freezing effect generally can not change because of patient's difference, and is less demanding to the monitoring of coagulation function.2. natural hirudin deactivation free zymoplasm not only, but also deactivation and scleroproein bonded zymoplasm are so it is more obvious than heparin to suppress the effect of thrombus growth.3. natural hirudin does not combine with the platelet factor VI, and good anticoagulation is still arranged near embolus, can not cause thrombopenia.4. natural hirudin anticoagulation and vitamin K are irrelevant, and the influence of unable to take food thing, and it is little to interact between the medicine.Because above-mentioned these advantages, as the natural hirudin of direct thrombin inhibitors, at present, and in Europe and north America region, the traditional heparin of recommended replacement, routine is used for the anti-freezing control of cardiovascular disorder.
Research is also arranged, and the pharmacologically active power of leech depends on that the antithrombin activity intensity of its activity in vivo composition is natural hirudin content, and the antithrombin activity that is to say leech is by force a natural hirudin content height, and its pharmacologically active is just strong, otherwise just.The antithrombin activity of hirudinaria manillensis obviously is better than the nonbloodsucking leech, the antithrombin activity composition of hirudinaria manillensis mainly is that natural hirudin is (English by name: Hirudin), natural hirudin exists only in the sialisterium and secreted saliva thereof of hirudinaria manillensis, and this has just increased difficulty for the extraction of natural hirudin.The antithrombin activity composition of nonbloodsucking leech does not contain natural hirudin, and up to the present, its activeconstituents is unclear as yet.
Production method retrieval to natural hirudin in the prior art is as follows:
1, Chinese patent: 03113566.8, denomination of invention: the cultural method of Hirudinaria manillensis and the extractive technique of r-hirudin.This invention is by introducing fine provenance or the open-air Hirudinaria manillensis that captures stalwartness, carry out artificial hierarchical segmented taming and dociling, feed, carry out artificial propagation, wait to raise to a certain degree, adopt chemical substance to stimulate fresh and alive Hirudinaria manillensis to make it secrete saliva, then the further separating and filtering of its saliva is obtained the r-hirudin crude product, again the r-hirudin crude product is carried out deep processing and obtain meeting medical r-hirudin, this Hirudinaria manillensis contains anticoagulative substance than other kind Hirudinaria manillensis content height, medicinal effect is good, and it is simple to extract the r-hirudin method, behind the extraction r-hirudin Hirudinaria manillensis is put back to the pond and cultures, be separated by one the week or can extract again again in 10 days, every fresh and alive Hirudinaria manillensis can extract 6-7 time repeatedly, can extract about r-hirudin crude product 6-7 milliliter good product quality at every turn, economize on resources again, improved the utility value of Hirudinaria manillensis.But what the technology that this invention provided was extracted only is the crude product of natural hirudin, and impurity is many, can not be directly for clinical use.
2, Chinese patent: 200610019531.0, denomination of invention: a kind of is the method and the application of feedstock production r-hirudin with the hirudinaria manillensis.This invention preparation method is characterised in that and comprises hirudinaria manillensis slurrying, preparation suspension liquid and dry three steps, resulting powder, and it has preserved the topmost effective active composition of hirudinaria manillensis---natural hirudin.Characteristics such as this invention has that technology is simple, production cost is lower, the product long preservative period, and can adopt Markwardt zymoplasm direct titrimetric method to detect natural hirudin content, thereby makes steady quality, and the product curative effect is guaranteed.R-hirudin has wide range of applications, can make capsule, soft capsule, granule, tablet use separately by the method for routine, or make compound preparation with other animal and plant medicine, be used to prevent and treat cardiovascular and cerebrovascular diseases such as cerebral apoplexy, hypertension, hyperlipidaemia, coronary heart disease.But should invention adopt spray drying device, its investment and floor space are all bigger, management inconvenience, and extraction yield is low, production cost is high.
3, Chinese patent: 200710066096.1, denomination of invention: a kind of extracting method of active ingredient of natural leech essence.This disclosure of the Invention a kind of extracting method of active ingredient of natural leech essence, be that water cleans the aqueous ethanolic solution that the clean back of hirudinaria manillensis adds content 15～25% and soaks after 30-50 hour, filter, the filtrate and the dregs of a decoction; The Hirudinaria manillensis dregs of a decoction are rubbed, and the aqueous acetone solution that adds content 15～25% again soaks, filter, filtrate, respectively twice filtrate vacuum reclaimed ethanol and acetone after, merging filtrate, conventional vacuum lyophilization obtains product.This invented technology is simple, with low cost, and yield is higher, good product quality, long preservative period, and whole process of production does not have " three wastes " discharging, and product is as the raw material in medicine, beauty treatment, protective foods three big fields.But in the natural hirudin leaching process, use ethanol, acetone, and ethanol and acetone are inflammable substance, exist potential safety hazard.
4, Chinese patent: 200710026462.0, denomination of invention: a kind of is raw-material technique for producing hirudin with the Hirudinaria manillensis frozen fresh body.This invention belongs to field of traditional Chinese medicine pharmacy, relates to the extraction of animal effective constituent, and purpose is to solve the problem that traditional r-hirudin extraction process is difficult to suitability for industrialized production.This process using is extracted r-hirudin from whole Hirudinaria manillensis, can suitability for industrialized production.Hirudinaria manillensis homogenate, soup compound normal saline extraction three times are filtered earlier.Filtrate is boiled, filtering and impurity removing albumen.Collect filtrate,, get the r-hirudin crude product behind the alcohol precipitation through freeze concentration.Again with physiological saline washing r-hirudin crude product, concentrated cleaning solution then, last lyophilize obtains product.This technique for producing hirudin method is simple, and final product quality is qualified, and The pharmacological results shows that the leech of extracting with this technology have great pharmacological effects.But in the natural hirudin leaching process, use ethanol, and ethanol is inflammable substance, exists potential safety hazard.
5, Chinese patent: 200710026461.6, denomination of invention: a kind of is raw-material technique for producing hirudin with Hirudinaria manillensis saliva.This invention belongs to field of traditional Chinese medicine pharmacy, relates to the extraction of animal effective constituent.Purpose is to solve the problem that traditional r-hirudin extraction process is difficult to suitability for industrialized production.This technology is extracted r-hirudin with Hirudinaria manillensis saliva, can suitability for industrialized production.Hirudinaria manillensis saliva is used normal saline extraction, and extracting solution boils, filters, gets filtrate.The solid substance normal saline extraction filters, and gets filtrate.Whole filtrates before and after merging add Lyphgel and handle twice, filter, and go filtrate.The solid substance normal saline extraction filters.Filtrate is boiled back cooling rapidly, and refrigeration is spent the night.Centrifugal, getting behind the supernatant liquor concentrating under reduced pressure again, lyophilize becomes lyophilized powder.This production technique is simple, and product vigor height has very strong thrombus dissolving effect.But the natural hirudin that this invention is extracted, its impurity is more, and purity is lower.
Summary of the invention
The objective of the invention is in order to solve above-mentioned problems of the prior art, a kind of extracting method of new natural hirudin is provided, obtain having the purity height, the measured natural hirudin of matter.
The present invention is achieved in that
The production method of natural hirudin is characterized in that: it comprises, and the extraction that contains natural hirudin liquid, acid are heavy, heating, dehydration and exsiccant technology, and concrete steps are as follows:
(1) contains the extraction of natural hirudin liquid: choose hirudinaria manillensis, directly put into the paste roller mill defibrination through removal of impurities, after cleaning, drying, make slurries, extraordinarily going into physiological saline or tap water by 1～10 of slurry weight then soaked 20～40 minutes, with getting its filtrate after the filtration of 40～150 eye mesh screens, standby; Perhaps extrude secretory product, get its filtrate after filtering with 40～150 eye mesh screens from the live body hirudinaria manillensis, standby;
(2) acid is heavy: in above-mentioned filtrate, add trichoroacetic acid(TCA), precipitate, leave standstill 10～60 minutes with pH to 0.5～3.5 of trichoroacetic acid(TCA) regulator solution, and centrifugal, remove throw out, get supernatant liquor, standby;
(3) heating: above-mentioned supernatant liquor is packed in the container, place 50～85 ℃ water bath with thermostatic control water proof heating 20～50 minutes, take out and be cooled to normal temperature, use pH to 6.0～8.0 of sodium hydrate regulator solution again, centrifugal, remove throw out, get supernatant liquor, standby;
(4) dehydration: take off the moisture of supernatant liquor 30%～80% in the step (3), get concentrated solution, standby;
(5) drying: add water-soluble filler in above-mentioned concentrated solution, its add-on is 0.1%～50% of a concentrated solution weight, carries out lyophilize then, obtains the powder finished product and is the natural hirudin product.
More than (2), (3) said centrifugal be centrifugal 5～20 minutes with 5000 rev/mins～15000 rev/mins whizzer.
Above-described hirudinaria manillensis is to contain the hirudinaria manillensis live body of natural hirudin or the corpse of its dry body or its freezing preservation.
Above-describedly extrude secretory product from the live body hirudinaria manillensis, be to allow the induced liquid that allows after the abundant hunger of hirudinaria manillensis its organic acid, mineral acid, inorganic salt with lower concentration of having enough be mixed with again, then it is pushed, make it secrete saliva and intravital induced liquid, standby, hirudinaria manillensis continues to raise.Concrete grammar is seen " extracting the method for natural hirudin from the Hirudinaria manillensis live body " of 38-2 Tai'an, Minzu Road ,Nanning City ,Guangxi Zhuang Autonomousnationality District mansion 18 Zhou Weihai of floor Guangxi Ke Kang biotech firm application of present patent application relating to persons, number of patent application: 03113566, Granted publication day: 20060906, see the document 1 that above background technology provides, concrete technical scheme is: hirudinaria manillensis is cleaned up, use induced liquid fed to appetite hirudinaria manillensis then, the induced liquid that is adopted is the lower organic acid of concentration, mineral acid, inorganic salt, select acetate for use, citric acid, arginine, Methionin, Gelucystine, sodium-chlor, Repone K, yellow soda ash, one or both of sodium phosphate or potassium primary phosphate, two or more, its concentration expressed in percentage by weight is 0.1～3%, pH is between 6～8, be configured to the aqueous solution, at first hunger and thirst again to after its half an hour of fully having enough to hirudinaria manillensis, it is pushed, make it secrete saliva and intravital inductive substance, every fresh and alive hirudinaria manillensis can repeat to extract 6～7 times, can extract about 6～7 milliliters of r-hirudin crude products at every turn.
More than the water-soluble filler that adds in the said concentrated solution of (6) step be wherein a kind of or wherein two or more mixture of starch, sucrose, soluble cellulose, N.F,USP MANNITOL, maltodextrin and lactose.
The resulting natural hirudin product of the present invention can be used for the raw material of food, protective foods, medicine or makeup, have anticoagulant and thrombolytic, improve blood circulation, effect such as enhance metabolism, be used to prevent and treat cardiovascular and cerebrovascular diseases such as apoplexy, coronary heart disease, hyperlipidemia etc. clinically.
Resulting powder finished product can adopt Markwardt zymoplasm direct titrimetric method to detect the content of its activeconstituents, and the content of its natural hirudin is 100～50000 international unit/grams.
Can combine with the zymoplasm specificity according to r-hirudin, make the zymoplasm inactivation, it is the principle of 1:1 in conjunction with ratio, can detect the content of natural hirudin with Markwardt zymoplasm direct titrimetric method.
The measure unit of r-hirudin is an international unit, represents with ATU, and the international unit of thrombin activity is NIH, during promptly 1 ATU equals and the r-hirudin amount of 1 NIH zymoplasm.
The natural hirudin detection method of content:
Get the about 1g of this product powder, the accurate title, decide, the accurate 0.9% sodium chloride solution 5ml that adds, fully stir, lixiviate 30 minutes, and jolting constantly, centrifugal, precision is measured supernatant liquor 100 μ l, putting the Tutofusin tris hydrochloride buffer of test tube (among the 10mm * 100ml), adding the 0.5%(ox) Fibrinogen (in coagulum) (gets 0.2mol/L tris solution 25ml and the about 40ml of 0.1mol/L hydrochloric acid soln, adds water to 100 ml, regulate pH value to 7.4) 200 μ l, shake up, put slowly drip every 1ml (in) contain 40 U(units) in the water-bath (37 ℃ ± 0.5 ℃) thrombin solution (per minute 5 μ l shake up gently while dripping) to solidifying, record consumes the volume of thrombin solution, is calculated as follows:
U＝C1V1/C2V2W
In the formula: the every 1g of U---contains thrombin activity unit, i.e. r-hirudin content, ATU/g;
The concentration of C1---thrombin solution, μ/ml;
The concentration of C2---need testing solution, g/ml;
V1---consumes the volume of thrombin solution, μ l;
The add-on of V2---need testing solution, μ l;
The W---sampling amount.
Above-mentioned natural hirudin detection method of content all is applicable to the natural hirudin to be the quality monitoring of the made various products of main raw material.
The invention has the beneficial effects as follows:
Technology is simple, extraction yield is high, production cost is low, product purity is high, long preservative period, can adopt Markwardt zymoplasm direct titrimetric method to detect its active component content, particularly the present invention adopted that extraction unlike the prior art, acid are heavy, heating, dehydration and exsiccant technology, thereby make quality more stable, easy to carry and use, can be food, protective foods, medicine or makeup safety, fine raw material are provided, in clinical application, according to different needs, the present invention can be made pointed product.
Embodiment
Embodiment one:
Get 15 kilograms of Hirudo japonica live bodies, remove impurity, wash with water clean, naturally dry after, be placed in the paste roller mill repeatedly defibrination 3 times, obtain the Hirudo japonica slurries; Add 150 kilograms in physiological saline then, soaked 40 minutes, get 158.5 kilograms of filtrates after filtering with 150 eye mesh screens; Add trichoroacetic acid(TCA) in above-mentioned filtrate, regulating filtrate pH to 0.5, to carry out acid heavy, left standstill 10 minutes, and with centrifugal 30 minutes of 6000 rev/mins of whizzers, disgorging, must supernatant liquor 151.3 kilograms; Above-mentioned supernatant liquor packed into carry out the water proof heating in the water-bath, after treating that continuing thermostat(t)ed water after the interior supernatant liquor temperature of pot reaches 85 ℃ boils 25 minutes, take out cooling, regulate pH to 7.3 with sodium hydroxide again, then with centrifugal 10 minutes of 10000 rev/mins of whizzers, remove throw out, get 147.6 kilograms of supernatant liquors; It is that 3000 daltonian ultra-filtration equipment carry out ultrafiltration that above-mentioned supernatant liquor is placed molecular weight, make its leach in the supernatant liquor behind 31.3% the moisture 49.6 kilograms of concentrated solutions; Carry out lyophilize after the tapioca (flour) of adding 10% in above-mentioned concentrated solution, obtain 5.06 kilograms of natural hirudin powder finished products, the content through adopting Markwardt zymoplasm direct titrimetric method to detect this product natural hirudin is the 915ATU/ gram.
Embodiment two:
Get 30 kilograms of the Hirudinaria manillensis corpse of freezing icing preservation, treat to put into after it dissolves naturally in the paste roller mill repeatedly defibrination 4 times, obtain the Hirudinaria manillensis slurries; In slurries, add 60 kilograms of clean waters then, soaked 25 minutes, get 80.8 kilograms of filtrates after filtering with 100 eye mesh screens; Add trichoroacetic acid(TCA) in above-mentioned filtrate, regulating pH to 3.3, to carry out acid heavy, left standstill 45 minutes, with centrifugal 5 minutes of 10000 rev/mins of whizzers, removes throw out, must supernatant liquor 71.1 kilograms; Above-mentioned supernatant liquor is put into the electrically heated jacketed kettle carry out the water proof heating, treat to continue thermostatically heating after 50 minutes after the interior supernatant liquor temperature of pot reaches 50 ℃, take out cooling, transfer the pH joint to 6.5 with sodium hydroxide again, then with centrifugal 15 minutes of 15000 rev/mins of whizzers, remove throw out, get 68.4 kilograms of supernatant liquors; With the above-mentioned supernatant liquor molecular weight of packing into is in the 5000 daltonian dialysis tubings, places distilled water, after dialysing 12 hours under 4 ℃ the condition, dialyzate; With 68.4 kilograms of polyoxyethylene glycol dialyzate is dewatered, make its take off in the dialyzate behind 79.3% the moisture 14.2 kilograms of concentrated solutions; In above-mentioned concentrated solution, add 0.142 kilogram medical starch and 0.142 kilogram maltodextrin respectively, and then carry out lyophilize, obtain 0.296 kilogram of natural hirudin powder finished product, the content through adopting Markwardt zymoplasm direct titrimetric method to detect this product natural hirudin is the 49500ATU/ gram.
Embodiment three:
Get 200 kilograms of Hirudinaria manillensis live bodies, remove impurity, wash with water clean, naturally dry after, be placed in the paste roller mill repeatedly defibrination 2 times, obtain the Hirudinaria manillensis slurries; Add 200 kilograms of clean waters then, soaked 30 minutes, get 363.7 kilograms of filtrates after filtering with 40 eye mesh screens; Add trichoroacetic acid(TCA) in above-mentioned filtrate, it is heavy that adjusting pH to 1.7 carries out acid, left standstill 60 minutes, centrifugal with 10000 rev/mins of tubular-bowl centrifuges, removes throw out, gets 332.2 kilograms of supernatant liquors; Above-mentioned supernatant liquor is packed in the Aluminum Drum, the water bath with thermostatic control that places 75 ℃ was then carried out poach 20 minutes after (reaching temperature with water-bath from the temperature of supernatant liquor begins to count when identical), take out cooling, transfer the pH joint to 7.0 with sodium hydroxide again, then centrifugal with 15000 rev/mins of tubular-bowl centrifuges, remove throw out, get 325.3 kilograms of supernatant liquors; It is that 4000 daltonian ultra-filtration equipment carry out ultrafiltration that above-mentioned supernatant liquor is placed molecular weight, make its leach in the supernatant liquor behind 53.7% the moisture 174.7 kilograms of concentrated solutions; Carry out lyophilize again add 20.5 kilograms N.F,USP MANNITOL in above-mentioned concentrated solution after, obtain 20.96 kilograms of natural hirudin powder finished products, the content through adopting Markwardt zymoplasm direct titrimetric method to detect this product natural hirudin is 4777 international unit/grams.
Embodiment four:
Get 50 kilograms of the Hirudo japonica corpse of freezing icing preservation, treat to put into after its self-dissolving in the paste roller mill repeatedly defibrination 3 times, obtain the Hirudo japonica slurries; Add 250 kilograms in physiological saline then, soaked 20 minutes, get 271.8 kilograms of filtrates after filtering with 80 eye mesh screens; Add trichoroacetic acid(TCA) in above-mentioned filtrate, regulating pH to 2.2, to carry out acid heavy, left standstill 20 minutes, with centrifugal 30 minutes of 7000 rev/mins of whizzers, removes throw out, must supernatant liquor 260.5 kilograms; Above-mentioned supernatant liquor is put into the temperature adjustment pot carry out the water proof heating, when reaching 85 ℃, the temperature of supernatant liquor counts constant temperature after 50 minutes, take out supernatant liquor and be cooled to normal temperature, then, transfer pH to 6.8 with sodium hydroxide, with centrifugal 15 minutes of 8000 rev/mins of whizzers, remove throw out, 258.6 kilograms of supernatant liquors; It is that 3000 daltonian ultra-filtration equipment carry out ultrafiltration that above-mentioned supernatant liquor is placed molecular weight, make it leach in the supernatant liquor 43.8% moisture after, 113.3 kilograms of concentrated solutions; The N.F,USP MANNITOL that adds 53.7 kilograms in above-mentioned concentrated solution carries out lyophilize then, obtains 54.16 kilograms of natural hirudin powder finished products, and the content through adopting Markwardt zymoplasm direct titrimetric method to detect this product natural hirudin is 356 international unit/grams.
Embodiment five:
5 kilograms of Hirudinaria manillensis live bodies of hungry 3 months of inventor leech feedlot are cleaned up, use 0.3% citric acid induced liquid fed to appetite Hirudinaria manillensis then, manually it is pushed after half an hour, to squeeze out its saliva and intravital induced liquid, altogether 12.6 kilograms of mixing solutionss, get 12.1 kilograms of filtrates after filtering with 150 eye mesh screens earlier, in above-mentioned filtrate, add trichoroacetic acid(TCA) then, it is heavy that adjusting pH to 3.0 carries out acid, left standstill 15 minutes, centrifugal with 15000 rev/mins of tubular-bowl centrifuges, remove throw out, get 10.3 kilograms of supernatant liquors; Above-mentioned supernatant liquor is packed in the Aluminum Drum, the water bath with thermostatic control that places 75 ℃ was carried out poach 40 minutes after (reaching temperature with water-bath from the temperature of supernatant liquor begins to count when identical), take out cooling, transfer the pH joint to 7.0 with sodium hydroxide again, then centrifugal with 15000 rev/mins of tubular-bowl centrifuges, remove throw out, get 9.6 kilograms of supernatant liquors; With the above-mentioned supernatant liquor molecular weight of packing into is in the 5000 daltonian dialysis tubings, places distilled water, after dialysing under 6 ℃ the condition 10 hours dialyzate; With 0.96 kilogram of polyoxyethylene glycol dialyzate is dewatered, make its take off in the dialyzate behind 30.2% the moisture 6.7 kilograms of concentrated solutions; The solubility tapioca (flour) that in above-mentioned concentrated solution, adds 3.35 kilograms, and then carry out lyophilize, obtain 3.42 kilograms of natural hirudin powder finished products, the content through adopting Markwardt zymoplasm direct titrimetric method to detect this product natural hirudin is 102 international unit/grams.
Embodiment six:
Get 10 kilograms of the Hubei ox leech corpse of freezing icing preservation, treat to put into after its self-dissolving in the paste roller mill repeatedly defibrination 3 times, obtain Hubei ox leech slurries; Add 30 kilograms in tap water then, soaked 35 minutes, get 36.7 kilograms of filtrates after filtering with 50 eye mesh screens; Add trichoroacetic acid(TCA) in above-mentioned filtrate, it is heavy that adjusting pH to 2.8 carries out acid, left standstill 25 minutes, centrifugal with 12000 rev/mins of separating machines, removes throw out, gets 32.4 kilograms of supernatant liquors; Above-mentioned supernatant liquor is put into the temperature adjustment pot carry out the water proof heating, when reaching 75 ℃, the temperature of supernatant liquor counts constant temperature after 45 minutes, take out supernatant liquor and be cooled to normal temperature, then, transfer pH to 6.8 with sodium hydroxide, with centrifugal 25 minutes of 8000 rev/mins of whizzers, remove throw out, 31.6 kilograms of supernatant liquors; It is that 4000 daltonian ultra-filtration equipment carry out ultrafiltration that above-mentioned supernatant liquor is placed molecular weight, make it leach in the supernatant liquor 53.4% moisture after, 14.73 kilograms of concentrated solutions; In above-mentioned concentrated solution, add 3.0 kilograms W-Gum and 3.63 kilograms lactose respectively, carry out lyophilize then, obtain 6.88 kilograms of natural hirudin powder finished products, the content through adopting Markwardt zymoplasm direct titrimetric method to detect this product natural hirudin is 636 international unit/grams.
Claims (7)
1. the production method of a natural hirudin is characterized in that: it comprises, and the extraction, the acid that contain natural hirudin liquid are heavy, heating, dehydration and exsiccant technology, and concrete steps are as follows:
(1) contains the extraction of natural hirudin liquid: choose hirudinaria manillensis, directly put into the paste roller mill defibrination through removal of impurities, after cleaning, drying, make slurries, extraordinarily going into physiological saline or tap water by 1～10 of slurry weight then soaked 20～40 minutes, with getting its filtrate after the filtration of 40～150 eye mesh screens, standby; Perhaps extrude secretory product, get its filtrate after filtering with 40～150 eye mesh screens from the live body hirudinaria manillensis, standby;
(2) acid is heavy: in above-mentioned filtrate, add trichoroacetic acid(TCA), precipitate, leave standstill 10～30 minutes with pH to 0.5～3.5 of trichoroacetic acid(TCA) regulator solution, and centrifugal, remove throw out, get supernatant liquor, standby;
(3) heating: above-mentioned supernatant liquor is packed in the container, place 50～85 ℃ water bath with thermostatic control water proof heating 20～50 minutes, take out and be cooled to normal temperature, use pH to 6.0～8.0 of sodium hydrate regulator solution again, centrifugal, remove throw out, get supernatant liquor, standby;
(4) dehydration: take off the moisture of supernatant liquor 30%～80% in the step (3), get concentrated solution, standby;
(5) drying: add water-soluble filler in above-mentioned concentrated solution, its add-on is 0.1%～50% of a concentrated solution weight, carries out lyophilize then, obtains the powder finished product and is the natural hirudin product.
2. the production method of natural hirudin according to claim 1, it is characterized in that: described hirudinaria manillensis is to contain the hirudinaria manillensis live body of natural hirudin or the corpse of its dry body or its freezing preservation.
3. the production method of natural hirudin according to claim 1, it is characterized in that: describedly extrude secretory product from the live body hirudinaria manillensis, be to allow allow after the abundant hunger of hirudinaria manillensis it have enough again with the acetate of lower concentration, citric acid, arginine, Methionin, Gelucystine, sodium-chlor, Repone K, yellow soda ash, wherein one or both of sodium phosphate or potassium primary phosphate, two or more, its concentration expressed in percentage by weight is 0.1～3%, the induced liquid of pH between 6～8, then it is pushed, make it secrete saliva and intravital induced liquid, standby, hirudinaria manillensis continues to raise.
4. the production method of natural hirudin according to claim 1 is characterized in that: step (2), step (3) described centrifugal be centrifugal 5～30 minutes with 5000 rev/mins～15000 rev/mins whizzer.
5. the production method of natural hirudin according to claim 1, it is characterized in that: it is that 3000～6500 daltonian ultra-filtration equipment directly dewater to supernatant liquor that the described dehydration of step (4) is to use the molecular weight of ultra-filtration membrane, the molecular weight of perhaps at first the supernatant liquor in the step (3) being packed into is in 3000～6500 daltonian dialysis tubings, place distilled water, under 4～10 ℃ condition, dialysed 6～12 hours, get dialyzate, then the polyoxyethylene glycol powder is sprinkling upon on the dialysis tubing surface, polyoxyethylene glycol adding weight is 0.1～1 times of dialyzate, make the dialyzate in the dialysis tubing take off 30%～80% moisture, get concentrated solution.
6. the production method of natural hirudin according to claim 1, it is characterized in that: the water-soluble filler that adds in the concentrated solution is wherein a kind of or wherein two or more mixture of starch, sucrose, soluble cellulose, N.F,USP MANNITOL, maltodextrin and lactose.
7. the product that obtains of the described natural hirudin production of claim 1 is as the purposes of food, protective foods, medicine or cosmetic material.
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Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102603890A (en) * | 2012-03-23 | 2012-07-25 | 韶关市颐林泉生物科技有限公司 | Method for extracting hirudin |
| CN102964446A (en) * | 2012-11-28 | 2013-03-13 | 周维官 | Method for repeatedly extracting natural hirudin from live vampire leech |
| CN104151425A (en) * | 2014-08-15 | 2014-11-19 | 周维海 | Large-scale production method for natural hirudin |
| CN105693851A (en) * | 2016-04-16 | 2016-06-22 | 徐顺青 | Hirudin extracting method |
| CN106266343A (en) * | 2016-08-31 | 2017-01-04 | 山东沃华医药科技股份有限公司 | A kind of preparation method of dispersing brain blood |
| CN106928347A (en) * | 2017-03-13 | 2017-07-07 | 佛山市爱的生物科技有限公司 | A kind of separation method of hirudin |
| CN107325177A (en) * | 2017-09-01 | 2017-11-07 | 荆州市民康生物科技有限公司 | A kind of method for improving Bufrudin extract yield |
| CN107337729A (en) * | 2017-07-25 | 2017-11-10 | 重庆多普泰制药股份有限公司 | A kind of thick extracting method of hirudin |
| CN107365381A (en) * | 2017-09-01 | 2017-11-21 | 荆州市民康生物科技有限公司 | A kind of method had children outside the state plan ripple auxiliary and improve Hirudo japonica element extract yield |
| CN107510705A (en) * | 2017-09-01 | 2017-12-26 | 荆州市民康生物科技有限公司 | A kind of freeze drying protectant of leech crude extract and lyophilized preservation technique |
| CN107661357A (en) * | 2017-11-16 | 2018-02-06 | 广西远程水蛭养殖有限公司 | Leech medicinal liquor preparation method |
| CN107827978A (en) * | 2017-09-30 | 2018-03-23 | 广西博白县琼达农业科技有限公司 | A kind of extracting method of hirudin |
| CN108578791A (en) * | 2018-04-24 | 2018-09-28 | 吴刚 | A kind of hirudin is modified the preparation method of anticoagulant material |
| CN108727487A (en) * | 2018-05-07 | 2018-11-02 | 广东海洋大学 | A kind of liquid film extraction method of hirudin |
| CN110623275A (en) * | 2019-09-18 | 2019-12-31 | 周维海 | Composite amino acid gilt-edged leech polypeptide food with anticoagulant and antithrombotic effects and preparation method thereof |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
| CN113101234A (en) * | 2021-03-30 | 2021-07-13 | 宁波博睿修存生物科技有限公司 | Hirudin active freeze-dried powder and preparation method and application thereof |
| CN113577821A (en) * | 2021-07-07 | 2021-11-02 | 三峡大学 | Repeated extraction device and method for hirudin in living body |
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| CN102603890A (en) * | 2012-03-23 | 2012-07-25 | 韶关市颐林泉生物科技有限公司 | Method for extracting hirudin |
| CN102964446A (en) * | 2012-11-28 | 2013-03-13 | 周维官 | Method for repeatedly extracting natural hirudin from live vampire leech |
| CN102964446B (en) * | 2012-11-28 | 2014-08-13 | 周维官 | Method for repeatedly extracting natural hirudin from live vampire leech |
| CN104151425A (en) * | 2014-08-15 | 2014-11-19 | 周维海 | Large-scale production method for natural hirudin |
| CN104151425B (en) * | 2014-08-15 | 2017-02-08 | 周维海 | Large-scale production method for natural hirudin |
| CN105693851A (en) * | 2016-04-16 | 2016-06-22 | 徐顺青 | Hirudin extracting method |
| CN106266343A (en) * | 2016-08-31 | 2017-01-04 | 山东沃华医药科技股份有限公司 | A kind of preparation method of dispersing brain blood |
| CN106928347A (en) * | 2017-03-13 | 2017-07-07 | 佛山市爱的生物科技有限公司 | A kind of separation method of hirudin |
| CN107337729A (en) * | 2017-07-25 | 2017-11-10 | 重庆多普泰制药股份有限公司 | A kind of thick extracting method of hirudin |
| CN107365381A (en) * | 2017-09-01 | 2017-11-21 | 荆州市民康生物科技有限公司 | A kind of method had children outside the state plan ripple auxiliary and improve Hirudo japonica element extract yield |
| CN107325177A (en) * | 2017-09-01 | 2017-11-07 | 荆州市民康生物科技有限公司 | A kind of method for improving Bufrudin extract yield |
| CN107510705A (en) * | 2017-09-01 | 2017-12-26 | 荆州市民康生物科技有限公司 | A kind of freeze drying protectant of leech crude extract and lyophilized preservation technique |
| CN107827978A (en) * | 2017-09-30 | 2018-03-23 | 广西博白县琼达农业科技有限公司 | A kind of extracting method of hirudin |
| CN107661357A (en) * | 2017-11-16 | 2018-02-06 | 广西远程水蛭养殖有限公司 | Leech medicinal liquor preparation method |
| CN108578791A (en) * | 2018-04-24 | 2018-09-28 | 吴刚 | A kind of hirudin is modified the preparation method of anticoagulant material |
| CN108727487A (en) * | 2018-05-07 | 2018-11-02 | 广东海洋大学 | A kind of liquid film extraction method of hirudin |
| CN108727487B (en) * | 2018-05-07 | 2021-05-07 | 广东海洋大学 | Liquid membrane extraction method of hirudin |
| CN110623275A (en) * | 2019-09-18 | 2019-12-31 | 周维海 | Composite amino acid gilt-edged leech polypeptide food with anticoagulant and antithrombotic effects and preparation method thereof |
| CN112480243A (en) * | 2021-01-06 | 2021-03-12 | 广西科康科技集团有限公司 | Large-scale hirudin separation and purification production process method and equipment |
| CN113101234A (en) * | 2021-03-30 | 2021-07-13 | 宁波博睿修存生物科技有限公司 | Hirudin active freeze-dried powder and preparation method and application thereof |
| CN113577821A (en) * | 2021-07-07 | 2021-11-02 | 三峡大学 | Repeated extraction device and method for hirudin in living body |
| CN113577821B (en) * | 2021-07-07 | 2022-08-30 | 三峡大学 | Repeated extraction device and method for hirudin in living body |
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